Measuring circulating therapeutic antibody, antigen and antigen/antibody complexes using ELISA assays

ABSTRACT

The present invention relates to the field of immunology and hyperproliferative diseases. More specifically, the present invention relates to a method of detecting and monitoring therapeutic antibody:antigen complex, soluble antigen and soluble therapeutic antibody, wherein a patient has undergone at least one course of immunotherapy. Yet further, levels of therapeutic antibody:antigen complexes, soluble antigens or soluble therapeutic antibodies may be measured and used to stage or monitor a hyperproliferative disease.

This application is a continuation of U.S. patent application Ser. No.14/854,753, filed Sep. 15, 2015, now abandoned which is a divisional ofU.S. patent application Ser. No. 14/305,430, filed Jun. 16, 2014, nowU.S. Pat. No. 9,157,916, which is a divisional of U.S. patentapplication Ser. No. 13/934,940, filed Jul. 3, 2013, now U.S. Pat. No.8,790,882, which is a divisional of U.S. patent application Ser. No.13/543,560, filed Jul. 6, 2012, now U.S. Pat. No. 8,501,423, which is adivisional of U.S. patent application Ser. No. 13/349,170, filed Jan.12, 2012, now U.S. Pat. No. 8,232,068, which is a divisional of U.S.patent application Ser. No. 13/080,180, filed Apr. 5, 2011, now U.S.Pat. No. 8,114,618, which is a continuation of U.S. patent applicationSer. No. 12/761,903, filed Apr. 16, 2010, now U.S. Pat. No. 7,943,332,which is a divisional of U.S. application Ser. No. 10/251,144, filed onSep. 20, 2002, now U.S. Pat. No. 7,718,387, which claims priority toU.S. Provisional Application No. 60/323,679, filed Sep. 20, 2001. Theentire text of each of the above referenced disclosures is specificallyincorporated herein by reference.

BACKGROUND OF THE INVENTION A. Field of Invention

The present invention relates to the field of immunology andhyperproliferative diseases. More particularly, the present inventionrelates to a method of detecting and monitoring a therapeuticantibody:antigen complex, soluble antigen, free therapeutic antibody andsoluble total therapeutic antibody, wherein a patient has undergone atleast one dose of immunotherapy. Yet further, the methods may be used tomonitor or stage a hyperproliferative disease by measuring the levels oftherapeutic antibody:antigen complexes, soluble antigens or solubletherapeutic antibodies.

B. Description of the Related Art

1. Clusters of Differentiation

Clusters of differentiation (CD) have been established to define humanleukocyte differentiation antigens (Bernanrd and Boumsell, 1984) by thecomparison of reactivities of monoclonal antibodies directed against thedifferentiation antigens. These cell surface antigens serve as markersof cell lineage and distinguish populations of leukocytes with differentfunctions, e.g., neutrophils and monocytes.

Leukocyte cell surface antigens have enormous clinical applicationpotential for the identification of leukocyte populations and theirfunctional status (Krensky, 1985, Kung et al., 1984; Kung et al., 1983;Cosimi et al., Knowles et al., 1983; and Hoffman, 1984). For example,measuring the total numbers of T cells by surface markers has beenuseful for the characterization, diagnosis and classification oflymphoid malignancies (Greaves, et al., 1981) and viral infectionassociated with transplantation (Colvin, R. B et al., 1981), and AIDS(Gupta, 1986; Ebert et al., 1985).

a) CD20

CD20, also called B1 (Bp35), is a cell surface phosphoprotein detectedon the surface of B-lymphocytes (Tedder and Schlossman, 1988; Warzynskiet al., 1994; Algino et al. 1996). CD20 has a major role in theregulation of human B-cell activation, proliferation and differentiation(Golay et al., 1985; Tedder and Engel, 1994; Kehrl et al., 1994). It hasbeen reported that CD20 is heavily phosphorylated in malignant B-cellsand proliferating B-cells when compared to non-proliferating B-cells(Tedder and Schlossman, 1988). Based on sequence analysis, the CD20molecule appears to have four transmembrane domains with n- andc-terminal domains in the cytoplasm (Kehrl, et al., 1994). The moleculeappears to regulate transmembrane Ca⁺⁺ conductance (Tedder and Engel,1994). Antibodies directed towards the extracellular portion of CD20appear to activate a tyrosine kinase pathway that modulates cell cycleprogression by interaction with src-related kinases (Deans et al., 1995;Popoff et al. 1998; Hofmeister et al., 2000). Relocalization of CD20into a detergent-insoluble membrane compartment upon binding toantibodies has also been reported (Deans et al., 1998). Severalinvestigators have documented variations in the intensity of CD20expression on the surface of malignant B-cells in differentlymphoproliferative diseases (Almasri et al., 1992; Ginaldi et al.,1998). This is important in view of the success an anti-CD20 monoclonalantibody (Rituximab) in treating various B-cell malignancies (Maloney etal., 1999; Dimopoulous et al., 2000; Zinzani et al., 2000; Hainsworth,2000; Keating et al., 2000; McLaughlin et al., 2000: Kuehnle et al.,2000). The reported structure of the CD20 molecule suggests that it isnot secreted and is highly unlikely to be shed from the cell surface(Riley et al., 2000).

b) CD52

The CD52 antigen is a glycoprotein with a very short mature proteinsequence consisting of 12 amino acids, but with a large carbohydratesdomain (approximately 3 times the size of the protein domain) (Xia, M.Q. et al., 1993; Treumann, A. et al., 1995). CD52 is expressed on thesurface of T- and B-lymphocytes, monocyte/macrophages, eosinophils andsome hematopoietic progenitors (Rowan, W. et al., 1998; Elsner, J. etal., 1996; Taylor, M. L. et al., 2000; Gilleece, M. H. et al., 1993).CD52 is also expressed in the male reproductive tract, mainly in theepithelial lining cells of the distal epidermis, vas deferens, andseminal vesicles (Kirchhoff, C. et al., 1995; Kirchhoff, C. et al.,1993; Kirchhoff, C. 1996; Kirchhoff, C. et al., 1997; Kirchhoff, C.,1998; Kirchhoff, C. et al., 2000). CD52 is necessary for spermatozoa topreserve normal motility. It is shed into seminal plasma and thenacquired by sperm cells to enable their passage through the genitaltract, thus it is detectable on the surface of epididymal sperm and inthe ejaculate, but not on either spermatogenetic cells or testicularspermatozoa. The protein core of the sperm and lymphocyte CD52 isidentical—both are products of a single copy gene located on chromosome1(1p36) (Tone, M. et al., 1999). However, N-linked carbohydrate sidechains and the GPI-anchor structure are different. The physiologicalrole of CD52 on lymphocytes is unclear.

The Campath-1 family of monoclonal antibodies was originally generatedby immunizing rats against human T-cells (Friend, P. J. et al., 1991).Later studies show that Campath-1 antibodies recognize CD52 (Xia, M. Q.et al., 1993; Xia, M. Q. et al., 1991; Hale, G. et al., 1990). Severalforms, both IgG and IgM, were generated. The IgG1 form of Campath-1 washumanized and this agent, Campath-1H (Alemtuzumab), has recently beenapproved for the treatment of refractory chronic lymphocytic leukemia(CLL) Finkelstein, J. B. et al., 2001; Rawstron, A. C. et al., 2001;Riechmann, L. et al., 1988). The Campath-1 family of antibodies is alsobeing used in vitro for lymphocyte depletion in allogeneic marrow graftsand is being investigated as immunomodulatory therapy in a variety ofdiseases (Moreau, T. et al., 1996; Matteson, E. L. et al., 1995; Lim, S.H. et al., 1993; Lockwood, C. M. et al., 1993; Lockwood, C. M., 1993;Lockwood, C. M. et al., 1996; Dick, A. D. et al., 2000; Hale, G. et al.,2000; Isaacs, J. D. et al., 1992; Lim, S. H. et al., 1993; Mehta, J. etal., 1997; Naparstek, E. et al., 1999; Naparstek, E. et al., 1995;Novitzky, N. et al., 1999; Or, R. et al., 1994).

Antibodies against CD52 are believed to initiate killing of cellsthrough antigen cross-linking (Hale, C. et al., 1996). As a result ofthis cross-linkage, several cytokines are released including tumornecrosis factor-α, interferon γ and interleukin (Elsner, J. et al.,1996; Wing, M. G. et al., 1996; Wing, M. G. et al., 1995). Cross-linkingof CD52 by antibodies promotes apoptosis and antibody-dependent cellularcytotoxicity, which may count for the effectiveness of Campath-1H intreating patients with chronic lymphocytic leukemia (CLL) (Rowan, W. etal., 1998; Rawstron, A. C. et al., 2001; Greenwood, J. et al., 1994;Xia, M. Q., et al., 1993). CD52 is expressed on the surface ofneoplastic lymphocytes in patients with CLL, low-grade lymphomas andT-cell malignancies (Dyer, M. J., 1999; Dybjer, A. et al., 2000; Pawson,R. et al., 1997; Salisbury, J. R. et al., 1994; Matutes, E. 1998). Somecases of myeloid, monocytic and acute lymphoblastic leukemia alsoexpress CD52 (Belov, L. et al., 2001; Hale, G. et al., 1985). This wideexpression of CD52 in a variety of hematological malignancies has led toincreasing interest in using Campath-1H in treating these malignancies(Khorana, A. et al., 2001; Keating, M. J. 1999).

CD52 is shed in the male productive system and the soluble moleculesplay an important role in preserving spermatozoa function (Kirchhoff,C., 1996; Yeung, C. H. et al., 1997; Yeung, C. H. et al., 2001).However, it is not known if CD52 is shed from hematopoietic cells and/ordetectable in the circulation of patients with CLL.

c) CD33

CD33 is a member of the siglecs family which bind sialic acid. CD33 isrestricted to the myelomonocytic lineage of cells. During maturation ofmyloid cells, the pluripotent hematopoietic stem cells give rise toprogenitor cells that have a diminished self-renewal capacity and agreater degree of differentiation. During this development, normalmyeloid cells express cell surface antigens, for example CD33. CD33 ispresent on maturing normal hematopoietic cells, however, normalhematopoietic stem cells lack this cell surface antigen. In addition tomaturing normal hematopoietic cells, CD33 is also present on acutemyelocytic leukemia (AML). Thus, this myeloid cell surface maker hasbecome an attractive target for monoclonal antibody targeting. Yetfurther, anti-CD33 antibodies have also been used to deliver radiationor a cytotoxic agent directly to leukemic cells.

2. Immunoassays

Immunoassays are usually used to measure cell surface antigens.Typically, immunofluorescence using flow cytometry is the immunoassay ofchoice. However, other immunoassays may be used, for example enzymelinked immunosorbant assays (ELISA). This technique is based upon thespecial properties of antigen-antibody interactions with simple phaseseparations to produce powerful assays for detecting biologicalmolecules.

One well-known and highly specific ELISA is a sandwich ELISA. In thisassay, the antibody is bound to the solid phase or support, which isthen contacted with the sample being tested to extract the antigen fromthe sample by formation of a binary solid phase antibody:antigencomplex. After a suitable incubation period, the solid support is washedto remove the residue of the fluid sample and then contacted with asolution containing a known quantity of labeled antibody.

The methodology and instrumentation for the ELISA is simpler than thatfor immunofluorescence. Yet further, the ELISA and immunofluorescenceassays are completely different assays. ELISA assays measure the protein(antigen) in the plasma/serum, which reflects the entire body. Surfaceimmunofluorescent assays measure an antigen on the surface of individualcells and does not provide information on the amount of cells in thebody. Thus, there are advantages in developing an ELISA assay to providea measurement of the entire body.

BRIEF SUMMARY OF THE INVENTION

It is, therefore, an objective of the present invention to providemethods for detecting or monitoring soluble leukocyte surface molecules,such as cell differentiation antigens or fragments thereof.Specifically, soluble surface antigen, antibody:antigen complexes andantibodies that are directed to cell differentiation antigens may bedetected or monitored by using a modified sandwich ELISA technique.Also, soluble cell surface molecules as quantified using the modifiedsandwich ELISA technique can be used to monitor proliferation and cellvolume in individuals with cancer or other hyperproliferation diseasesdue to any other process, such as inflammation or infection.

In specific embodiments, the antibody:antigen complex is measured in apatient that has undergone at least one course, e.g., an injection, ofimmunotherapy with a therapeutic antibody. The therapeutic antibody mayinclude, but is not limited to anti-CD20, anti-CD52 or anti-CD33. Theantibody:antigen complex is measured by ELISA techniques and provides adetermination of the efficacy of the antibody immunotherapy.

Another aspect of the present invention includes a method of providingan immunotherapy to a patient comprising administering to the patient atherapeutic antibody and detecting the presence of a circulatingantibody:antigen complex, total antibody, free antigen and freeantibody. The therapeutic antibody binds to a soluble antigen, which isshed from the cell surface. It is envisioned that the antigen is CD20,CD52 and CD33. It is envisioned that these methods can be used tomonitor the efficacy of antibody-based therapy.

In further embodiments, the present invention provides methods fordetecting or monitoring hyperproliferative diseases by measuring solubleleukocyte surface molecules, therapeutic antibodies, or antibody:antigencomplexes. Specifically, a sample from a patient is obtained, the sampleis contacted with a first monoclonal antibody, in which the antibodycaptures the complex; the complex is contacted with a labeled secondantibody; and the labeled complex is measured. The first monoclonalantibody is bound to a solid surface. Yet further, the patient hasundergone a course of immunotherapy with a therapeutic antibody in whichthe therapeutic antibody binds to a soluble circulating target antigenforming the complex.

Hyperproliferative disease as used herein may be further defined ascancer. Yet further, cancer is further defined as a neoplasm. Exemplaryneoplasms include, but are not limited to melanoma, non-small cell lung,small-cell lung, lung hepatocarcinoma, retinoblastoma, astrocytoma,gliobastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast,pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma,cervical, gastrointestinal, lymphoma, brain, colon, or bladder.

It is also contemplated that hyperproliferative disease may be furtherdefined as an autoimmune disease, for example, but not limited toSjögren's syndrome, rheumatoid arthritis, systemic lupus erythematosus,autoimmune thyroid disease, refractory ocular inflammatory disease,multiple sclerosis, Wegener's granulomatosis or infection.

In specific embodiments, the present invention monitors, detects orstages a hematopoietic neoplasm. Exemplary hematopoietic neoplasms,include, but are not limited to chronic lymphocytic leukemia, acutemyelogenous leukemia, acute lymphoblastic leukemia, myelodysplasticsyndrome, chronic myelomonocytic leukemia, juvenile myelomonocyteleukemia, multiple myeloma, lymphoma, T-cell chronic lymphocyticleukemia or prolymphocytic leukemia.

Yet further, it is contemplated that the present invention may be usedto determine tumor mass. Tumor mass may be determined using the modifiedsandwich ELISA of the present invention to measure the levels of solubleleukocyte cell surface antigens, soluble antibodies or solubleantibody:antigen complexes.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows a Western blot demonstrating levels of sCD20 in the plasmaof chronic lymphocytic leukemia (CLL) patients. Protein extract fromnormal peripheral blood mononuclear (MN) cells show no expression ofCD20 while leukemic cells from patients with CLL (C) show the expectedproteins with molecular weight of 33-36 KD. The plasma from normalindividuals (NP) as well as from patients with CLL show the soluble CD20(sCD20).

FIG. 2 shows a Western blot demonstrating levels of sCD52 in the plasmaof CLL patients. Protein extract from leukemic cells (C) show theexpected proteins and the plasma from the same patient (P) show thesoluble sCD52. CD52 is also detected in protein extract from peripheralblood mononuclear cells from normal individual (NC) as well as in normalplasma (NP).

FIG. 3 shows higher levels of sCD20 in patients with CLL as comparedwith normal individuals.

FIG. 4 illustrates the linearity of sCD52 determined by ELISA.

FIG. 5 illustrates that CLL patients have higher levels of sCD52 ascompared with normal individuals.

FIG. 6 shows that the sCD20/Rituximab complexes increased with anincrease in the levels of Rituximab.

FIG. 7 shows the direct correlation between sCD20 levels and Raistaging.

FIG. 8 shows a direct correlation between sCD20 levels and Binetstaging.

FIG. 9 shows the median survival of patients with high sCD20 compared topatients with low sCD20.

FIG. 10 shows that the levels of sCD52 correlate with Rai staging.

FIG. 11 shows the levels of sCD52 correlate with Binet staging.

FIG. 12 illustrates that higher levels of sCD52 are detected in CLLpatients with poor cytogenetics.

FIG. 13 illustrates that higher levels of sCD52 are detected in patientswith higher number of lymph node sites with enlarged lymph nodes.

FIG. 14 illustrates that patients with high levels of sCD52 have ashorter survival than patients with low levels of sCD52.

FIG. 15 shows the detection of the sCD52/Campath-1H complexes in a CLLpatient being treated with Campath-1H for minimal residual. Levels ofsCD52 are also shown.

DETAILED DESCRIPTION OF THE INVENTION

The concept of using antibodies in treating cancers orhyperproliferative diseases depend on the ability of the antibodies toreach the tumor cells that express the antigen to which the antibodybinds. The presence of free soluble target antigen in the circulationand the possibility of binding and absorbing these antibodies by thecell-free or soluble antigen, and thus preventing these antibodies fromreaching the malignant cells, is of concern of the present invention.

The present invention as described herein utilizes therapeuticantibodies to diagnose, monitor or stage hyperproliferative diseases andefficacy of therapy. More particularly, the present invention relates toa method of detecting and monitoring a therapeutic antibody:antigencomplex, soluble antigen and soluble therapeutic antibody, wherein apatient has undergone at least one course of immunotherapy.

A variety of hyperproliferative diseases can be monitored, staged ordiagnosed according to the methods of the present invention. Ahyperproliferative disease includes disease and conditions that areassociated with any sort of abnormal cell growth or abnormal growthregulation. Hyperproliferative disease may be further defined as cancer.Yet further, cancer may be defined as a neoplasm or tumor. Exemplaryneoplasms that may be monitored or diagnosed using the present inventioninclude, but are not limited to melanoma, non-small cell lung,small-cell lung, lung hepatocarcinoma, retinoblastoma, astrocytoma,gliobastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast,pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma,cervical, gastrointestinal, lymphoma, brain, colon, or bladder.

More particularly, the methods of the present invention may be used tomonitor, stage or diagnose a hematopoietic neoplasm, for example, butnot limited to chronic lymphocytic leukemia, acute myelogenous leukemia,acute lymphoblastic leukemia, myelodysplastic syndrome, chronicmyelomonocytic leukemia, juvenile myelomonocyte leukemia, multiplemyeloma, lymphoma, T-cell chronic lymphocytic leukemia, prolymphocyticleukemia, lymphomas, B cell related diseases or other T cell relateddiseases.

Other hyperproliferative diseases contemplated for diagnosing, stagingor monitoring are Sjögren's syndrome, rheumatoid arthritis, systemiclupus erythematosus, autoimmune thyroid disease, refractory ocularinflammatory disease, multiple sclerosis, Wegener's granulomatosis andpre-neoplastic lesions in the mouth, prostate, breast, or lung.

It is also envisioned that a combination of soluble markers, solubleantibodies or soluble antibody:antigen complexes may be measured tomonitor, stage or diagnosis a hyperproliferative disease. For example,but not limited to, it is envisioned that soluble anti-CD20/CD20 andsoluble anti-CD52/CD52 complexes may be measured simultaneously orconsecutively to monitor, stage or diagnosis a hyperproliferativedisorder.

The presence of circulating target antigens and possible consequentformation of circulating immune complexes may be important fortherapeutic approaches based on using antibodies against specificantigens, including Campath-1H (anti-CD52) (Hale et al., 2000; Khoranaet al., 2001; Flynn, 2000) or Mylotarg (anti-CD33), (Van Der Vleden etal., 2001) which are also used to treat patients with leukemias. It iscontemplated that soluble antigens may bind to the therapeutic antibodyin patients receiving the therapeutic antibody to form immune complexes,i.e., antibody:antigen complexes. These immune complexes may reduce theamount of therapeutic antibody from reaching the hyperproliferatingcells. Thus, the dosages of the therapeutic antibodies may need to beadjusted accordingly to reach therapeutic levels, such that the antibodyis able to reach the target cell having the antigen. Thus, it also iscontemplated that the measurement of soluble cell surface antigens andits complexes with therapeutic antibodies may help in designing moreeffective therapeutic strategies.

Yet further, it is contemplated that the present invention may be usedto determine tumor mass. Tumor mass may be determined using the modifiedsandwich ELISA described herein to measure the levels of solubleleukocyte cell surface antigens, soluble antibodies or solubleantibody:antigen complexes.

A. Cell Surface Antigens

As used herein “target antigen”, “surface antigen”, “cell surfaceantigen”, or “leukocyte cell surface antigen” are interchangeable andsimply refer to the cell surface antigen which is located on theleukocyte, e.g., CD20, CD52, or CD33.

The presence of circulating target antigens and possible consequentformation of circulating immune complexes may be important for othertherapeutic approaches based on using antibodies against specificantigens, including, but not limiting to Rituximab (anti-CD20),Campath-1H (anti-CD52) or Mylotarg (anti-CD33).

1. CD20

CD20 is an important molecule in the maturation and proliferation ofCD20 positive B-cells (Riley, J. K. et al., 2000). Marked differences inthe intensity of CD20 expression in various B-cell malignancies suggestthat CD20 may be associated with the different clinical behaviors of thelymphoproliferative disorders (Marti, G. E. et al., 1992 and Ginaldi, L.et al., 1998). It is contemplated that soluble CD20 (sCD20) can bedetected in the plasma of both normal individuals and patients with CLL.Yet further, it is contemplated in the present invention that sCD20 maybe due to active shedding or to turn-over of cells and fragmentation ofthe cell membrane or both.

sCD20 may be assessed in plasma, cell lysate or serum. In specificembodiments, sCD20 may be assessed in plasma rather than serum to reducethe risk of the clotting process damaging circulating cells andinfluencing the levels of sCD20.

The levels of sCD20 may have a direct impact on patients' management andprognosis. sCD20 may play an important role when patients are treatedwith anti-CD20 (Rituximab). The formation of sCD20/Rituxiamb complexesmay reduce the amount of monoclonal antibody that reaches the leukemiccells. If this is a factor, the dosages of the antibodies may need to beadjusted accordingly to reach therapeutic levels, particularly inpatients with high levels of sCD20.

2. CD52

Human CD52 (Campath-1H antigen) is an abundant surface molecule onlymphocytes and an important target for therapy of variouslymphoproliferative disorders (Tone, M. et al., 1999; Keating, M. J. etal., 1999; Kalil, N. et al., 2000). It comprises a smallglycosylphosphatidylinositol (GPI) anchored peptide to which a largecarbohydrate moiety is attached (Kirchhoff, C. et al., 2001).

It is contemplated by the inventor that CD52 may shed from cells in afashion similar to that reported in the male productive system(Kirchhoff, C. et al., 2001). It also is possible that in patients withCLL, the reticuloendothelial system is unable to remove all the cellulardebris that result from the turn-over of cells despite the relativelylow turn-over rate.

Soluble CD52 (sCD52) may be assessed in plasma, cell lysate or serum. Inspecific embodiments, sCD52 may be assessed in plasma rather than serumto reduce the risk of the clotting process damaging circulating cellsand influencing the levels of sCD52.

Yet further, it also is contemplated in the present invention that thepresence of sCD52 may have significant impact on the effectivenessand/or toxicities of Campath-1H therapy. CD52 is considered to be a veryappropriate target for monoclonal antibody therapy because of itsabundant expression on target cells, its close apposition to the targetcell membrane, and low rate of modulation (Treumann, A. et al., 1995;Dyer, M. J. et al., 1999; Bindon, C. I. et al., 1988). The response toCampath-1H is not uniform and the causes of this variability have beenexamined by a number of investigators. It has been suggested that thisvariability may partially depend on differences in the level of CD52expression on target cells (Ginaldi, L. et al., 1998).

3. CD33

CD33 is restricted to the myelomonocytic lineage of cells. CD33 ispresent on maturing normal hematopoietic cells, however, normalhematopoietic stem cells lack this cell surface antigen. In addition tomaturing normal hematopoietic cells, CD33 is also present on acutemyelocytic leukemia (AML). Thus, this myeloid cell surface maker hasbecome an attractive target for monoclonal antibody targeting.

Soluble CD33 (sCD33) may be assessed in plasma, cell lysate or serum. Inspecific embodiments, sCD33 may be assessed in plasma rather than serumto reduce the risk of the clotting process damaging circulating cellsand influencing the levels of sCD33.

B. Antibodies

The present invention is directed to the measurement of solubleleukocyte cell surface antigens, soluble antibodies to leukocyte cellsurface antigens, or soluble antibody/antigen complexes, and the use ofsuch measurements in the diagnosis and therapy of diseases anddisorders.

As used herein, the term “soluble” refers to those molecules that are“spontaneously released”; i.e., released by normal or pathologicphysiological processes of the cell, and those molecules present insoluble form in a body fluid by virtue of their in vivo administrationto the patient. Such molecules are to be distinguished from“solubilized” cell surface forms of the molecules, whose solubilizationis brought about by in vitro manipulation such as cell lysis bydetergent. The soluble leukocyte cell surface antigens of the inventionare molecules which carry antigenic determinants of their cell-surfacecounterparts.

The measurement of the soluble molecules of the invention can bevaluable in monitoring the effect of a therapeutic treatment on apatient, detecting and/or staging a disease in a patient and indifferential diagnosis of the physiological condition of a subject.These measurements can also aid in predicting therapeutic outcome and inevaluating and monitoring the immune status of patients. More than onetype of soluble molecule can be measured. The soluble molecules can bemeasured in any body fluid of the subject including, but not limiting toserum, plasma, urine, saliva, pleural effusions, synovial fluid, spinalfluid, tissue infiltrations and tumor infiltrates.

In certain aspects of the invention, one or more of the antibodies maybe a commercially available therapeutic antibody. For example, but notlimited to, Rituximab (anti-CD20), Campath-1H (anti-CD52) or Mylotarg(anti-CD33). These antibodies may be used in various diagnostic ortherapeutic applications, described herein below.

Yet further, it is also contemplated that one or more antibodies may beproduced to the cell surface antigens CD20, CD52 and CD33. It will beunderstood that polyclonal or monoclonal antibodies specific for theCD20, CD52 and CD33 and related proteins will have utilities in severalapplications. These include the production of diagnostic kits for use indetecting and diagnosing hyperproliferative disease.

1. Polyclonal Antibodies

Polyclonal antibodies to the CD20, CD52 and CD33 receptors generally areraised in animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the CD20, CD52 and CD33 receptors and an adjuvant.

Animals are immunized against the immunogenic composition orderivatives. Animals are boosted until the titer plateaus. The animalsare usually bled through an ear vein or alternatively by cardiacpuncture. The removed blood is allowed to coagulate and then centrifugedto separate serum components from whole cells and blood clots. The serummay be used as is for various applications or else the desired antibodyfraction may be purified by well-known methods, such as affinitychromatography using another antibody, a peptide bound to a solidmatrix, or by using, e.g., protein A or protein G chromatography.

2. Monoclonal Antibodies

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep, goat, monkey cells also is possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The animals are injected with antigen, generally as described above forpolyclonal antibodies. The antigen may be coupled to carrier moleculessuch as keyhole limpet hemocyanin if necessary. The antigen wouldtypically be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster injections with the same antigen wouldoccur at approximately two-week intervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens or lymph nodes. Spleen cells and lymph node cells arepreferred, the former because they are a rich source ofantibody-producing cells that are in the dividing plasmablast stage.

Often, a panel of animals will have been immunized and the spleen ofanimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1976), and those using polyethylene glycol (PEG),such as 37% (v/v) PEG. The use of electrically induced fusion methodsalso is appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, infusedcells (particularly the infused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways.

A sample of the hybridoma can be injected (often into the peritonealcavity) into a histocompatible animal of the type that was used toprovide the somatic and myeloma cells for the original fusion (e.g., asyngeneic mouse). Optionally, the animals are primed with a hydrocarbon,especially oils such as pristane (tetramethylpentadecane) prior toinjection. The injected animal develops tumors secreting the specificmonoclonal antibody produced by the fused cell hybrid. The body fluidsof the animal, such as serum or ascites fluid, can then be tapped toprovide MAbs in high concentration.

The individual cell lines could also be cultured in vitro, where theMAbs are naturally secreted into the culture medium from which they canbe readily obtained in high concentrations.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the purified monoclonal antibodiesby methods which include digestion with enzymes, such as pepsin orpapain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate monoclonals. For this, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and control cellse.g., normal-versus-tumor cells. The advantages of this approach overconventional hybridoma techniques are that approximately 10⁴ times asmany antibodies can be produced and screened in a single round, and thatnew specificities are generated by H and L chain combination whichfurther increases the chance of finding appropriate antibodies.

3. Humanized Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen.

4. Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described in the art (Kozbor,1984; Brodeur, et al., 1987).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. (Jakobovits et al., 1993).

Alternatively, the phage display technology (McCafferty et al., 1990)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.

5. Antibody Conjugates

The present invention further provides antibodies to CD20, CD52 and CD33or another secondary antibody for example but not limited to goatanti-human IgG, generally of the monoclonal type, that are linked to atleast one agent to form an antibody conjugate. In order to increase theefficacy of antibody molecules as diagnostic or therapeutic agents, itis conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one effector or reporter molecule. Effectormolecules comprise molecules having a desired activity, e.g., cytotoxicactivity. Non-limiting examples of effector molecules which have beenattached to antibodies include toxins, anti-tumor agents, therapeuticenzymes, radio-labeled nucleotides, antiviral agents, chelating agents,cytokines, growth factors, and oligo- or poly-nucleotides. By contrast,a reporter molecule is defined as any moiety which may be detected usingan assay. Non-limiting examples of reporter molecules which have beenconjugated to antibodies include enzymes, radiolabel s, haptens,fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an antibody conjugate. Such properties may beevaluated using conventional immunological screening methodology knownto those of skill in the art. Sites for binding to biological activemolecules in the antibody molecule, in addition to the canonical antigenbinding sites, include sites that reside in the variable domain that canbind pathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic oranti-cellular agent, and may be termed “immunotoxins”.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody-directed imaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present invention may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the invention may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a SEPHADEX® (cross-linked dextran gel) column and applying theantibody to this column. Alternatively, direct labeling techniques maybe used, e.g., by incubating pertechnate, a reducing agent such asSNC12, a buffer solution such as sodium-potassium phthalate solution,and the antibody. Intermediary functional groups which are often used tobind radioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeALEXA FLUOR® 350, ALEXA FLUOR® 430, AMCA, BODIPY® 630/650, BODIPY®650/665, BODIPY®-FL, BODIPY®-R6G, BODIPY®-TMR, BODIPY®-TRX, CASCADEBLUE®, CY®3, CY®5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, OREGONGREEN® 488, OREGON GREEN® 500, OREGON GREEN® 514, PACIFIC BLUE™, REG,Rhodamine Green, Rhodamine Red, RENOGRAPHIN®, ROX, TAMRA, TET,Tetramethylrhodamine, and/or TEXAS RED® (red fluorescent dye).

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and/or avidinand streptavidin compounds. The use of such labels is well known tothose of skill in the art and are described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

C. Immunodetection Methods

In certain embodiments, the present invention concerns immunodetectionmethods for binding, purifying, removing, quantifying and/or otherwisegenerally detecting biological components such as soluble CD20, solubleCD52, soluble CD33, anti-CD22, anti-CD52, anti-CD33, anti-CD20/CD20,anti-CD52/CD52, and anti-CD33/CD33. Some immunodetection methods includeenzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay,bioluminescent assay, and Western blot to mention a few. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Doolittle M H and Ben-Zeev O,1999; Gulbis B and Galand P, 1993; De Jager R et al., 1993; and Nakamuraet al., 1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a therapeutic antibody:antigen complex, e.g.,anti-CD20/CD20, anti-CD52/CD52 and anti-CD33/CD33, or a solubleleukocyte cell surface antigen e.g., CD20, CD52 or CD33, or a solubleantibody, e.g., anti-CD20, anti-CD52, or anti-CD33, contacting thesample with a first monoclonal antibody which captures the complex, andcontacting the sample with a composition capable of selectively bindingor detecting the complex, e.g., a labeled second antibody, underconditions effective to allow the formation of immunocomplexes. Otherexamples of compositions capable of selectively binding or detecting thecomplex include, but are not limited to a antibodies or other ligandsthat can be labeled using a variety of markers, e.g., biotin/avidinligand binding arrangement, as is known in the art. One skilled in theart may also use a labeled third antibody.

In terms of antigen, antibody or antibody:antigen complex detection, thebiological sample analyzed may be any sample that is suspected ofcontaining an antigen or antibody:complex, such as, for example, atissue section or specimen, a homogenized tissue extract, a cell, anorganelle, separated and/or purified forms of any of the aboveantigen-containing compositions, or even any biological fluid that comesinto contact with the cell or tissue, including blood and/or serum,although tissue samples or extracts are preferred.

Contacting the chosen biological sample with the first antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, anyanti-CD20/CD20, anti-CD52/CD52 and anti-CD33/CD33 complex or anyantigens present i.e., CD20, CD52 or CD33, or any antibodies presenti.e., anti-CD20, anti-CD52 or anti-CD33. After this time, thesample-antibody composition, such as a tissue section, ELISA plate, dotblot or western blot, will generally be washed to remove anynon-specifically bound antibody species, allowing only those antibodiesspecifically bound within the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art. Allprior assays to detect immunocomplexes are based on autologous complexesgenerated by the patients own antibodies and antigen. The presentinvention is different in that the assays of the present inventiondetect immunocomplexes as a result of a therapeutic approach.

The antigen, antibody or antigen:antibody complex employed in thedetection may itself be linked to a detectable label, wherein one wouldthen simply detect this label, thereby allowing the amount of theprimary immune complexes in the composition to be determined.Alternatively, the first antibody that becomes bound within the primaryimmune complexes may be detected by means of a second binding ligandthat has binding affinity for the antibody. In these cases, the secondbinding ligand may be linked to a detectable label. The second bindingligand is itself often an antibody, which may thus be termed a“secondary” antibody. The primary immune complexes are contacted withthe labeled, secondary binding ligand, or antibody, under effectiveconditions and for a period of time sufficient to allow the formation ofsecondary immune complexes. The secondary immune complexes are thengenerally washed to remove any non-specifically bound labeled secondaryantibodies or ligands, and the remaining label in the secondary immunecomplexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousdiseases wherein a leukocyte cell surface antigens are shed during thedisease process, or therapeutic antibodies accumulate in the circulationor therapeutic antibodies form complexes. Here, a biological and/orclinical sample suspected of containing soluble leukocytes cell surfaceantigens, e.g., CD20, CD52, CD33, soluble therapeutic antibodies, e.g.,anti-CD20, anti-CD52, anti-CD33 or therapeutic antibody:antigencomplexes e.g., anti-CD20/CD20, anti-CD52/CD52 or anti-CD33/CD33 aremeasured.

In the clinical diagnosis and/or monitoring of patients with variousforms a hyperproliferative disease, such as, for example, cancer, thealteration in the levels of a soluble leukocytes markers, solubletherapeutic antibodies and soluble therapeutic antibody:antigencomplexes in comparison to the levels in a corresponding biologicalsample from a normal subject is indicative of a patient with cancer orother hyperproliferative diseases. However, as is known to those ofskill in the art, such a clinical diagnosis would not necessarily bemade on the basis of this method in isolation. Those of skill in the artare very familiar with differentiating between differences in typesand/or amounts of biomarkers, which represent a positive identification,and/or low level and/or background changes of biomarkers. Indeed,background levels are often used to form a “cut-off” above whichincreased detection will be scored as significant and/or positive.

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, antibodies of the leukocyte cell surface antigen(e.g., anti-CD20, anti-CD33, anti-CD52) are immobilized onto a selectedsurface exhibiting protein affinity, such as a well in a polystyrenemicrotiter plate. Then, a sample from a patient that has undergone atleast one course, e.g., one injection, of immunotherapy with atherapeutic antibody, e.g., Rituxiamb, Campath-H or Mylotarg is added tothe wells. After binding and/or washing to remove non-specifically boundimmune complexes, the bound therapeutic antibody:antigen complex may bedetected. Detection is generally achieved by the addition of secondantibody that is linked to a detectable label. This type of ELISA is asimple “sandwich ELISA”.

In another exemplary sandwich ELISA used to detect the therapeuticantibody, the antibody of the leukocyte cell surface antigen isimmobilized onto the well surface. Next, cell lysate from a patient thathas undergone at least one course, e.g., one injection, of immunotherapywith a therapeutic antibody is added to the wells. The cell lysatecontains an antigen that binds to the antibody. Then, plasma from thepatient is added to the well. The plasma contains the therapeuticantibody which will bind to the antigen. After binding and/or washing toremove non-specifically bound immune complexes, the bound therapeuticantibody:antigen complex may be detected. Detection is achieved by theaddition of a third antibody that is linked to a detectable label.

In yet another exemplary sandwich ELISA used to detect the leukocyteantigen, the antibody of the leukocyte cell surface antigen isimmobilized onto the well surface. Next, plasma from a patient that hasundergone at least one course, e.g., one injection, of immunotherapywith a therapeutic antibody is added to the wells. After binding and/orwashing to remove non-specifically bound immune complexes, the boundtherapeutic antibody:antigen complex may be detected. Then, a secondantibody that is linked to a detectable label is added. The secondantibody is typically the therapeutic antibody which has been labeled.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

It also is contemplated that the above reagents maybe packaged in a kitthat may be produced commercially to measure the soluble antigens,antibodies or antibody:antigen complexes described herein.

D. Immunotherapy

In specific embodiments of the present invention, it is provided thatthe patient has undergone at least one course, e.g., one injection, ofimmunotherapy. As described herein, immunotherapeutics, generally, relyon the use of immune effector cells and molecules to target and destroycancer cells. The immune effector may be, for example, an antibodyspecific for some marker on the surface of a tumor cell. The antibodyalone may serve as an effector of therapy or it may recruit other cellsto actually effect cell killing. The antibody also may be conjugated toa drug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells.

In specific embodiments, the antibody that is used for the immunotherapyis Rituximals, Campath-1H, or Mylotarg.

Immunotherapy could also be used as part of a combined therapy. In oneaspect of immunotherapy, the tumor cell must bear some marker that isamenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155. An alternative aspect of immunotherapy is tocombine pro-apoptotic effect with immune stimulatory effects. Immunestimulating molecules also exist including: cytokines such as IL-2,IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8and growth factors such as FLT3 ligand.

As discussed earlier, examples of immunotherapies currently underinvestigation or in use are immune adjuvants (e.g., Mycobacterium bovis,Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds)(U.S. Pat. No. 5,801,005; U.S. Pat. No. 5,739,169; Hui and Hashimoto,1998; Christodoulides et al., 1998), cytokine therapy (e.g., interferonsα, β and γ; IL-1, GM-CSF and TNF) (Bukowski et al., 1998; Davidson etal., 1998; Hellstrand et al., 1998) gene therapy (e.g., TNF, IL-1, IL-2,p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. No.5,830,880 and U.S. Pat. No. 5,846,945) and monoclonal antibodies (e.g.,anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998;Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Herceptin(trastuzumab) is a chimeric (mouse-human) monoclonal antibody thatblocks the HER2-neu receptor. It possesses anti-tumor activity and hasbeen approved for use in the treatment of malignant tumors (Dillman,1999).

1. Passive Immunotherapy

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow.

Preferably, human monoclonal antibodies are employed in passiveimmunotherapy, as they produce few or no side effects in the patient.However, their application is somewhat limited by their scarcity andhave so far only been administered intralesionally. Human monoclonalantibodies to ganglioside antigens have been administeredintralesionally to patients suffering from cutaneous recurrent melanoma(Irie & Morton, 1986). Regression was observed in six out of tenpatients, following, daily or weekly, intralesional injections. Inanother study, moderate success was achieved from intralesionalinjections of two human monoclonal antibodies (Irie et al., 1989).

It may be favorable to administer more than one monoclonal antibodydirected against two different antigens or even antibodies with multipleantigen specificity. Treatment protocols also may include administrationof lymphokines or other immune enhancers as described by Bajorin et al.(1988). The development of human monoclonal antibodies is described infurther detail elsewhere in the specification.

2. Active Immunotherapy

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogenic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranath& Morton, 1991; Morton & Ravindranath, 1996; Morton et al., 1992;Mitchell et al., 1990; Mitchell et al., 1993). In melanomaimmunotherapy, those patients who elicit high IgM response often survivebetter than those who elicit no or low IgM antibodies (Morton et al.,1992). IgM antibodies are often transient antibodies and the exceptionto the rule appears to be anti-ganglioside or anticarbohydrateantibodies.

3. Adoptive Immunotherapy

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated anigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro. This form of immunotherapy hasproduced several cases of regression of melanoma and renal carcinoma,but the percentage of responders were few compared to those who did notrespond.

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

E. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Patient Samples

Plasma samples were collected from patients with CLL and normalindividuals. Peripheral blood samples were collected in EDTA tubes. Thediagnosis of CLL was established based on morphologic, immunologic andmolecular evaluation of peripheral blood and bone marrow. Immunologicevaluation included flow cytometric analysis of leukemic cells usingCD19, CD5, CD20, CD23, CD11C, CD22, FMC-7, CD79B, CD3, CD4, CD8, kappaand lambda. Molecular studies included immunoglobulin and T-cellreceptors genes as well as Bcl-1 and Bcl-2 rearrangement studies.

Example 2 Western Blot Analysis of Plasma and Cellular CD20

Five microliter of plasma from normal and CLL patients wereelectrophoretically separated on 9.5 SDS-PH gels. Cell lysates fromnormal mononuclear cells and CLL cell samples were used as positive andnegative controls. The nitrocellulose membrane was blocked with 5%non-fat milk in PBS containing 0.1% TWEEN® 20 and 0.01% sodium azide for6-8 hours at room temperature. The blots were incubated overnight at 4°C. with 1 μg/μ1 mouse anti-CD20 antibody (Sigma Chemical Corporation,St. Louis, Mo.) and PBS containing 2.5% non-fat milk, 2.5 bovine serumalbumin (BSA) and 0.1% TWEEN® 20. The membrane was then washed with PBScontaining 0.1% TWEEN® 20. The blot was then incubated with 1:20 dilutedanti-mouse horseradish peroxidase-conjugated Ig (Sigma ChemicalCorporation, St. Louis, Mo.) and PBS containing 1% non-fat milk and 0.1%TWEEN® 20. Immunoreactive bands were developed using the ECL detectionsystem (Amersham, Arlington Heights, Ill.).

As shown in FIG. 1, reactive bands with anti-CD20 were detected in theplasma of patients with CLL at high levels. The detected soluble (sCD20)bands in the plasma correspond to the 35 kD CD20 that was detected inCLL cells. Plasma from normal individuals also showed low levels ofsCD20. CLL cells showed easily detectable CD20 protein, but mononuclearcells from normal individuals, which are richer with monocytes andT-cells and contain few B-cells, showed no detectable CD20 bands.

Example 3 Western Blot Analysis of Plasma and Cellular CD52

Exactly 14.5 ul of total plasma from patients and 40 microgram ofcellular protein were electrophoresed on 9.5 SDS-PAGE gels. Cell lysatesfrom normal mononuclear cells and CLL cell samples were used as positiveand negative controls. The protein was transferred into nitrocellulosemembranes using standard techniques. Membranes were blocked with 5%non-fat milk in PBS containing 0.1% TWEEN®-20 and 0.01% of sodium azidefor 6 to 8 hours at warm temperature. The blots were incubated overnightat 4° C. with 1 ug anti-CD52 (Campath-1G) antibodies and PBS containing2.5 non-fat milk, 2.5 bovine serum albumin (BAS) and 0.01 TWEEN® 20. Themembranes were then washed with PBS containing 0.01% TWEEN® 20. Theblots were then incubated with TWEEN® 20 diluted goat anti-rat Ig linkedto horseradish peroxidase (Sigma Chemical Corporation, St. Louis, Mo.)and PBS containing 1% non-fat milk and 0.1% TWEEN® 20. Radio-activebands were developed using ECL detection system (Amersham, ArlingtonHeights, Ill.).

As shown in FIG. 2, protein extracts from leukemic cells show theexpected 14 to 20 kD CD52 glycoproteins as detected using Campath-1Gmonoclonal antibody. Plasma from the same patient showed correspondingbands.

Example 4 ELISA Analysis of Plasma and Cellular CD20

An ELISA assay for detecting sCD20 in the plasma of patients wasdeveloped. Briefly, a 96-well polystyrene microplate was coated withcapturing antibody for CD20 purchased from Sigma. Plates were thenwashed 6 times with PBS containing 0.01% TWEEN® 20, blocked with BSA inPBS containing 0.01% TWEEN® 20 for 1 to 3 hours at 37° C., washed in PBScontaining 0.01% TWEEN® 20. 100 microliter of plasma was added to thewells. The mixture was then incubated at room temperature for two hours.The sCD20 was detected using humanized anti-CD20 (Rituximab) antibodyafter horseradish peroxidase-enzyme conjugation using standardtechnique; Rituximab was diluted 1:400 in 2% BSA 0.01% TWEEN® 20. Theplates were incubated for twelve hours. The wells were then washed 6times with PBS containing 0.01% TWEEN® 20. 100 units of substrate wereadded for the development of the color and incubated for 15 to 30minutes with constant shaking. The reaction was then stopped with 15microliters of sodium chloride, the plates were read at 450 nmwavelength. Serial dilution of known number of molecules of syntheticCD20 peptide was used to generate a standard curve.

The intensity of the CD20 bands on the Western blot correlated with thelevels seen on the ELISA assay as shown in FIG. 1. Dilutions andmeasurements of diluted samples showed almost identical values. Levelsof sCD20 in the plasma of CLL patients as detected by ELISA assay weresignificantly higher than those detected in 31 normal individuals. (FIG.3). sCD20 levels in patients with CLL varied from 52.89 to 15740 M/ml(median=776.9). In contrast the levels of sCD20 in the plasma of normalindividuals varied between 123.55 to 547.10 M/ml (median=470).

Example 5 SCD52 Enzyme Linked Immunoabsorbant (ELISA) Assay

An ELISA assay for detecting the sCD52 in the plasma of patients wasdeveloped. Briefly, 96-well polystyrene microtiter plates were coatedwith Campath-1M antibodies. Plates were then washed 6 times with PBScontaining 0.01% TWEEN® 20, blocked with BSA in PBS containing 0.01%TWEEN® 20 for 1 to 3 hours at 37° C., washed in PBS containing 0.01%TWEEN® 20. 100 ul of patient's plasma was added and incubated for 3hours, then washed 8 times with PBS containing 0.01% TWEEN® 20. ThesCD52 was detected using the humanized anti-CD52 Campath-1H afterhorseradish peroxides labeling using standard techniques; the Campath-1Hwas diluted 1:400 in 2% BSA 0.01% TWEEN® 20. The wells were then washed6 times with PBS containing 0.01% TWEEN® 20. 100 units of substrate wereadded for the development of the color and incubated for 15 to 30minutes with constant shaking. The reaction was then stopped with 15microliters of sodium chloride, the plates were read at 450 nmwavelength.

The levels detected in the ELISA were normalized to those detected in 25normal individuals. The median level detected in the 25 normalindividuals was assigned a value of 1 and the levels detected in theplasma of CLL patients are expressed as folds of the normal median ofnormals. As shown in FIG. 2, the ELISA levels appear to correlate withthe levels detected on the western blot. The Western blot bands werescanned and quantified. Equal amounts of plasma from all samples wererun on the gel. In order to verify the linearity of the ELISA, dilutionsof plasma from a patient with high level and correlated the measurementswith dilutions. As shown in FIG. 4, there was complete correlationbetween the dilutions and the levels detected by the ELISA. As shown inFIG. 5, upon comparing the sCD52 levels detected in the normal patientswith those in CLL patients there was significant increase in the levelsof CD52 in CLL patients.

Example 6 Plasma CD20/Rituximab Complexes (ELISA)

sCD20/Rituximab complexes formation was investigated in the plasma ofCLL patients treated with Rituximab using an ELISA assay.

The plasma CD20/Rituximab complexes were measured using a similarsandwich ELISA assay. Briefly, a 96-well polystyrene microplate wascoated with capturing antibody for CD20 and washed as described above.Plasma samples were added after 1:100 dilution in PBS and incubated asdescribed above. For detection, goat anti-human immunoglobulin that washorseradish peroxidase conjugated was used. The wells were then washed 6times with PBS containing 0.01% TWEEN® 20. 100 units of substrate wereadded for the development of the color and incubated for 15 to 30minutes with constant shaking. The reaction was then stopped with 15microliters of sodium chloride, the plates were read at 450 nmwavelength. Serial dilution of known number of molecules of syntheticCD20 peptide after binding at saturation to Rituximab was used togenerate a standard curve.

sCD20/Rituximab immune complexes were detected in all 20 single samplesfrom 20 CLL patients being treated with Rituximab. The ELISA assayshowed linear correlation between dilutions of known amount of syntheticpeptide mixed with excess Rituximab (R=1). Immune complexes weredetected in serial samples from a patient being treated with Rituximab(FIG. 6). As shown in FIG. 6, sCD20/Rituximab complexes increased withan increase in the levels of the Rituximab.

Example 7 High Levels of sCD20 Correlate with Advanced Stage of CLL

The correlation of plasma levels of sCD20 in 180 CLL patients withvarious characteristics and clinical stages of the disease was assessed.The characteristics of the patients studied are listed in Table 1.

Thirty two (17.8%) of the patients had Rai stage 0 disease, 81 (45%)stage I-II, and 60 (33%) in stage III-IV. The median age of the patientswas 61 and median level of (32M was 3.4. There was no significantdifference in sCD20 levels between males and females (p-value=0.66).sCD20 levels were highly correlated with (32M (r=0.23, p-value=0.006),platelet count (r=−0.22, p-value=0.004), percentage of CD19+/CD38+ cells(r=0.20, p-value=0.03) and hemoglobin level (r=−0.18,p-value=0.02)(Table 2). sCD20 levels did not significantly correlatewith white blood count (r=−0.07, p-value=0.33), lymphocyte count(r=−0.03, p-value=0.71) or age (r=0.05, p-value=0.53). There was adirect correlation between sCD20 levels and Rai stages. When cases weregrouped as Rai 0, Rai I-II, and Rai higher Rai stages had significantlyhigher levels of sCD20 (P=0.01, Kruskal-Wallis test) (FIG. 7). Patientsin Rai stages 0-II had significantly lower sCD20 levels as compared topatients with Rai stages III-IV (P=0.01). Similar results were obtainedwhen Binet staging was used (P=0.004) (FIG. 8). There was no correlationbetween sCD20 levels and number of sites of lymphadenopathy (P=0.11) orhepatomegaly (P=0.25).

TABLE 1 Patient Characteristics (N = 180) Variable Median (range) N (%)Sex Male 116 (65) Female 63 (35) Rai 0 32 (17.8) 1 51 (29.5) 2 30 (16.7)3 15 (8.3) 4 45 (25.0) Parameter (median/range) Age in years 61 (33~84)CD38+/CD19+ (%) 7.7 (0.3~98) Hemoglobin g/dl 12.8 (4~17.8) Platelets ×10³/ul 142 (4~342) WBC × 10³/ul 55.9 (1.4~333.9) Lymphocytes (%) 85(9~99) β₂M mg/L 3.4 (1.3~12.7) sCD20 M/ml 776.9 (52.89~15740)

TABLE 2 Correlation of sCD20 with clinical parameters in patients withCLL Variable P-value (R-value) Hgb 0.02 (−0.18) β2m 0.006 (0.23) RAI (0,I-II, III-IV) 0.01 (Kruskal-Wallis) Binet (A, B, C) 0.004(Kruskal-Wallis) Platelets 0.004 (−0.22) % CD19+/CD38+ 0.03 (0.20)Splenomegaly 0.07 Hepatomegaly 0.25 Node Enlargement 0.11 Age 0.53(0.05) Lymphocytes 0.71 (−0.03) WBC 0.33 (−0.07) Sex 0.66

TABLE 3 Univariate Cox Proportional Hazards Model (N = 180) VariableCoefficient Relative Risk P-value Hemoglobin −0.241 0.79 0.0005 Log(β₂M)2 7.37 0.0005 RAI 0.353 1.42 0.018 RAI = 3, 4 (vs 0, 1, 2) 0.995 2.710.021 Platelets −0.008 0.99 0.020 Log(CD38+/CD19+) 0.551 1.73 0.023sCD20 0.0002 1.00 0.006 Log(sCD20) 0.374 1.45 0.080 sCD20 >= 1875 1.113.03 0.020 Age 0.025 1.02 0.22 Log(%-lymphocytes) 0.24 1.27 0.32Log(WBC) 0.183 1.2 0.43 Sex = male −0.13 0.88 0.76

Univariate Cox proportional hazards model was fitted (Table 3) to testfor variable that predicts survival in this patient group. As shown inTable 3, the expression of CD38, hemoglobin, platelets, β2M, Raistaging, and sCD20 were all predictors of survival. sCD20, as acontinuous variable in Cox regression model, was a predictor of survival(P=0.002). However, in light of the martingale residual plot forgoodness of fit, log transformation was needed in order to fit sCD20better to the model. When the logarithmic of sCD20 was used to re-fitthe univariate Cox model, it was only marginally significant withp-value 0.08. Therefore sCD20 was dichotomized using a cut off point of1875 M/ml chosen by CART, which demonstrated two groups of patients withsignificantly different survival profiles. Patients with sCD20>1875 M/mlhad significantly shorter survival times than those with sCD20<=1875M/ml (P=0.01) (FIG. 9). Median survival in the patients with high sCD20was approximately 18 months while the median in those with lower sCD20levels had not been reached (FIG. 9). Multivariate analysis showed thatthis shorter survival in patients with sCD20>1875 M/ml was independentof Rai stage or hemoglobin.

The levels of sCD20 may have a direct impact on patients' management andprognosis. The levels of sCD20 correlated directly with Rai and Binetstages, and 82M and negatively with platelets and hemoglobin. sCD20 didnot correlate with white cell count, age, splenomegaly, or lymph nodeenlargement. High levels of sCD20 correlated with shorter survivalindependently from the Rai staging. This suggested that sCD20 levels mayreflect a specific clinical stage of the disease as well a specificbiology. sCD20 may play an important role when patients are treated withanti-CD20 (Rituximab). The formation of sCD20/Rituxiamb complexes maysignificantly reduce the amount of monoclonal antibody from reaching theleukemic cells. If this is a factor, the dosages of the antibodies mayneed to be adjusted accordingly to reach therapeutic levels,particularly in patients with high levels of sCD20. The measurement ofsCD20 and its complexes with therapeutic antiCD20 antibodies may help indesigning more effective therapeutic strategies.

Example 10 Clinical Relevance of Soluble CD52 in CLL Patients

sCD52 levels were studied in 116 CLL patients. The characteristics ofthese patients are listed in Table 1. Of the 116 patients 66 (57%) werepreviously untreated. Seventy-nine (68%) were males and 44 (38%) were instage III to IV of Rai stage. The median age of the patient was 61 andthe median white-cell count was 60.1×10⁹/L. Median hemoglobin was 12.7g/l and the median β-microglobulin (132M) was 3.6 g/dL. Upon correlatingthe plasma levels of CD52 with Rai staging, there was significantcorrelation (p=0.0008, Kruskal-wallis) (FIG. 10). Also, there was asimilar correlation when Binet Staging (p<0.0001)(FIG. 11) was used. Ofthe 116 patients, 74 had complete cytogenetic studies and sCD52 levelswere compared between patients with poor cytogenetics (11q21-, +12, orabnormality on chromosome 17) and patients with other patients. Patientswith poor cytogenetic had significantly higher levels of sCD52(p=0.0002, kruskal-wallis) (FIG. 12). Plasma CD52 levels also correlatedwith the number of lymph node sites with enlarged lymph node. As shownin FIG. 13 higher levels of sCD52 were detected in the plasma ofpatients with higher number of lymph node sites with enlarged lymphnodes (P=0.002). sCD52 levels also positively correlated with increasingsize of liver (P=0.0005) and spleen (0.000002) (Table 5). There wasnegative correlation between sCD52 levels and hemoglobin (P<0.00001) andplatelets (P<0.00001) (Table 5). sCD52 levels directly correlated withtotal white cell count (WBC) (p<0.00001), β₂M (P=0.00002), and surfaceexpression of CD38 (0.01) (Table 5). Using Cox proportional hazard modeland univariate analysis, sCD52 strongly correlated with survival. Higherlevels of sCD52 correlated with shorter survival (p=0.001). Univariateanalysis also showed that survival in this patient group correlated withRai staging (p=0.007), surface CD38 expression (0.02), poor cytogenetics(p=0.005), hemoglobin (p=0.001), platelets (p=0.01), and β₂M (0.00001).Thus, the data suggested that this group of patients was representativeof CLL patients in general.

In multivariate analysis incorporating sCD52, Rai staging, hemoglobin,platelets, WBC, and β₂M, only β₂M was predictive of survival (p=0.02)while sCD52 was not predictive of survival. A cut-off point was used toseparate the patients into two groups, one with high expression and onewith low expression. Upon using a cut-off point of 32, which correspondto the upper quartile, patients with high levels of sCD52 showedsignificantly shorter survival (p=0.0001, Log-Rank test) (FIG. 14).Using sCD52 levels to separate CLL patients into two groups in amultivariate analysis incorporating Rai staging, hemoglobin, platelets,WBC, and β₂M, it was found that β₂M remained a predictor of survival(p=0.05) and sCD52 was a borderline predictor of survival (P=0.06),while Rai, hemoglobin, platelets, and WBC were not predictors ofsurvival. This suggested that sCD52 levels were possibly independentpredictor of survival when they were at very high levels.

TABLE 4 Patients Characteristics. Characteristic Patients (%) Male 79(68) Rai III-IV 44 (38) No prior Rx 66 (37) Splenomegaly 43 (37) Median(range) Age 61 (34-84) WBC × 10³/ul 60.4 (1.4-333) HGB 12.7 (4.0-17.8)B2M mg/L 3.6 (1.3-11.5)

TABLE 5 Spearman correlation between sCD52 levels and various clinicalcharacteristics in CLL. R p-value Hepatomegly (cm)   .31 .0005Splenomegly (cm)   .43 .000002 HGB −.45 .000000 PLT −.39 .000008 WBC  .53 .000000 LYM   .21 .01 β2M   .41 .00002 CD38/CD19   .44 .01 TNF-α  .38 .0003 AGE   .08 .4

These data demonstrated that sCD52 levels reflected specific clinicalbehavior in CLL. sCD52 levels correlated with various stages of thedisease and higher levels were associated with aggressiveness of thedisease. There was a direct correlation between the number of leukemiccells in circulation, hepatomegaly, splenomegaly, lymph nodeinvolvement, and 132M; all of which were usually associated with moreadvanced disease. Therefore, sCD52 can be used for staging patients, butmore importantly the presence of sCD52 may have significant impact onthe effectiveness of therapy of these patients when they are treatedwith Campath-1H. The possibility that sCD52 may bind to the therapeuticCampath-1H antibodies and sequester them from reaching the cells shouldbe considered and investigated. This binding may have significant impacton the pharmakodynamics and pharmakokenetics of the antibodies. It ispossible that patients with high levels of sCD52 require higher dosagesof antibodies to saturate the sCD52 and allow the antibodies to reachcells. Similarly, it is possible that patients on therapy will requirelower levels of antibodies as their sCD52 levels decrease and the tumormass become smaller. Reducing dosages of anti-CD52 antibodies may helpin reducing the severe immunsuppression reported in patients treatedwith Campath-1H. Using sCD52 may also be useful for monitoring the CLLdisease and monitoring the effectiveness of therapy, irrespective if itis based on Campath-1H or not.

Example 11 Detection of sCD52/Campath-1H Complexes in Patients Treatedwith Campath-1H

The detection of sCD52 in the plasma of patients with CLL led theinventor to investigate the possibility that antigens and antibodies mayform complexes in the plasma.

Plasma sCD52/Campath-1H complexes were measured using a similar sandwichELISA assay. Briefly, a ninety-six well polystyrene microplate wascoated with capturing antibody for CD52 and washed as described above.Plasma samples were added after 1:100 dilution in PBS and incubated asdescribed above. For detection, goat anti-human immunoglobulin that washorseradish peroxidase conjugated was used. The wells were then washed 6times with PBS containing 0.01% TWEEN® 20. 100 units of substrate wereadded for the development of the color and incubated for 15 to 30minutes with constant shaking. The reaction was then stopped with 15microliters of sodium chloride, the plates were read at 450 nmwavelength. Serial dilution of known number of molecules of syntheticCD52 peptide after binding at saturation to Campath-1H was used togenerate a standard curve.

sCD52/Campath-1H immune complexes were detected in samples from CLLpatients treated with Campath-1H. The ELISA assay showed linearcorrelation between dilutions of known amount of synthetic peptide mixedwith excess Campath-1H (R=1). Immune complexes were detected in serialsamples from a patient being treated with Campath-1H (FIG. 15).

The detection of sCD52/Campath-1H complexes in patients treated withCampath-1H suggests that the formation of these complexes were capableof reducing the amount of antibody available to attach to target cells.

Example 12 Correlation Between Campath-1H and Response to Therapy in CLL

Campath-1H levels were measured in patients with CLL treated withCampath-1H to eradicate minimal residual disease after chemotherapy. Allpatients were either in complete remission (CR), but had flow cytometryevidence of residual disease, or in partial remission (PR). The patientswere treated with Campath-1H 10 mg three times a week for one month. Theplasma levels of Campath-1H were correlated with residual disease asdetermined by polymerase chain reaction (PCR), response to therapy, andinfection.

Briefly, plasma samples were collected from 12 patients at the end ofthe course. Some patients achieved complete remission (CR) and somepatients had no response (NR). Patients with CR had significantly higherlevels of Campath-1H as compared to NR patients (P=0.009). The medianplasma Campath-1H level in patients achieving CR was 0.420 μg/ml (range0-1.760 μg/ml), while all NR patients had no detectable plasmaCampath-1H. Patients achieving CR had significantly lower minimaldisease as detected by PCR (P=0.02). Patients who started on therapywith more residual disease as detected by PCR had less probability ofachieving CR (P=0.02). Patients with evidence of infection (3 with CMVand one with Staphylococcus) had significantly more residual disease(P=0.02) as determined by PCR, but these patients had no statisticallysignificant difference in Campath-1H levels compared to those who didnot develop infection.

Thus, this data suggested that CLL patients with higher levels ofresidual disease may require higher doses of Campath-1H to eradicatedisease and detectable plasma levels of Campath-1H may be necessary forachieving CR. Furthermore infection in this group of patients wasassociated with higher levels of residual disease and not higher levelsof Campath-1H.

Example 13 Correlation Between Rituximab Levels and Response in Patientswith CLL Treated with Fludarbine, Cyclophosphamide and RituximabCombination

Treating CLL patients with fludarbine (F), cyclophosphamide (C) andRituximab (R) combination (Six cycles of FCR (F-25 mg/m2/day and C-250mg/m2/day on days 2-4 of cycle 1 and on days 1-3 of cycles 2-6 and R-375mg/m2 on day 1 of cycle 1 and R-500 mg/m2 on day 1 of cycles 2-6) hasresulted in success, such as achieving remission (CR).

Briefly, plasma Rituximab levels were measured in patients with CLLtreated with FCR at various time between 3 and 9 months of initiatingtherapy and the levels were correlated with response and othercharacteristics. When front-line patients were considered, patients whohad no response, had no detectable levels of Rituximab. These patientshad significantly higher levels of circulating CD20 (cCD20) as comparedwith patients with CR or nodular CR (CRN). However, when previouslytreated patients were considered, Rituximab levels were notsignificantly different between patients in CR, NR, and CRN. HigherRituximab levels were achieved in patients who were in earlier Rai stageat the time of initiating therapy (P=0.005) in front-line patients, butnot in previously treated patients (P=0.7). The achieved Rituximablevels correlated negatively with before therapy β₂M (R=−0.44, P=0.002)in previously treated patients, but not in front-line patients (R=0.20,P=0.23).

Thus, this data suggested that the few front-line patients treated onFCR, who did not respond to therapy, had low levels of Rituximab and maybenefit from higher doses.

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The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method for determining a dosage of atherapeutic anti-CD20 antibody for treating B cell hyperproliferativedisease, the method comprising testing a sample from a human to measurethe level of soluble CD20, and determining the dosage of the therapeuticanti-CD20 antibody according to the level of soluble CD20.
 2. The methodof claim 1, wherein said sample is selected from the group consisting ofplasma, serum, and cell lysate.
 3. The method of claim 1, wherein saidtherapeutic anti-CD20 antibody is Rituximab.
 4. The method of claim 1,wherein said B-cell hyperproliferative disease is chronic lymphocyticleukemia, acute lymphoblastic leukemia, myelodysplastic syndrome,chronic myelomonocytic leukemia, juvenile myelomonocyte leukemia,multiple myeloma, hairy cell leukemia, prolymphocytic leukemia, orlymphoma.
 5. The method of claim 4, wherein said B-cellhyperproliferative disease is chronic lymphocytic leukemia.
 6. Themethod of claim 1, further comprising administering said therapeuticanti-CD20 antibody to said human.
 7. The method of claim 3, furthercomprising administering said Rituximab to said human.